Reporter assays, in which the expression of an inert protein is driven by gene regulatory elements such as promoters and enhancers, are a workhorse for investigating gene regulation. Techniques for measuring reporter gene expression vary from single-cell or single-molecule approaches having low throughput to bulk Luciferase assays that have high throughput. We developed a Luciferase Reporter Assay using Flow-Cytometry (LucFlow), which measures reporter expression in single cells immunostained for Luciferase. We optimized and tested LucFlow with a murine cell line that can be differentiated into neutrophils, into which promoter-reporter and enhancer-promoter-reporter constructs have been integrated in a site-specific manner. The single-cell measurements are comparable to bulk ones but we found that dead cells have no detectable Luciferase protein, so that bulk assays underestimate reporter expression. LucFlow is able to achieve a higher accuracy than bulk methods by excluding dead cells during flow cytometry. Prior to fixation and staining, the samples are spiked with stained cells that can be discriminated during flow cytometry and control for tube-to-tube variation in experimental conditions. Computing fold change relative to control cells allows LucFlow to achieve a high level of precision. LucFlow, therefore, enables the accurate and precise measurement of reporter expression in a high throughput manner.
Copyright: © 2023 Nooti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.